Process for production of streptomycin



Patented Oct. 16, 1951 PROCESS FOR PRODUCTION OF STREPTOMYCIN Eugene L. Dulaney, Rahway, N. J., assignor to Merck & Co'., Inc., Rahway, N. J., a corporation of New Jersey No Drawing. Application July 25, 1950, Serial No. 175,891

1 Claim. 1

This invention relates to the production of streptomycin, and more particularly, to procedures for obtaining streptomycin in enhanced yield by the utilization of the new and distinct culture of Streptomyces griseus hereinafter described and characterized.

In my pending application, Serial No. 8,308, now U. S. Patent 2,545,572, filed February 14, 1 948, I have disclosed procedures for producing streptomycin in yields of the order of'800 to 1100 meg/ml. by means of the new and distinct culture S. griseus Dulaney L-118. This culture was obtained by subjecting spores of a strain of S. grz'seus characterized as resistant to an initial streptomycin concentration of at least 500 mcg./ml. to the action of ultra-violet light, allowing the spores surviving such treatment to grow and selecting from the resulting colonies of S. griseus those exhibiting an increased ability to produce streptomycin, again subjecting spores from the selected colonies to the action of ultraviolet light and repeating the colony develop ment, selection, and spore irradiation until a mutated strain of S. griseus was obtained which possessed the capacity of consistently producing streptomycin in yields of at least 800 mcg./ml.

While the streptomycin yields of 800 to 1100 meg/ml. produced by the culture S. griseus Dulaney L-118 are markedly superior to yields obtainable with strains of S. griseus previously available, I have now discovered that yields of streptomycin can be further increased to about 1800 to 2200 meg/ml. by utilizing the new and distinct culture hereinafter designated as S. griseus, albus mutant (Dulaney Z-38). This new culture or mutant was obtained by subjecting spores of S. grz'seus Dulaney L-118 to the mutating action of ultra-violet light and soft X-rays. The experimental procedure leading to the isolation of the new mutant was as follows:

Spores of S. grz'seus Dulaney L-l18 were washed from a surface growth with sterile distilled water, and the resulting spore suspension was filtered through several layers of sterile absorbent cotton. The filtered spore suspension was then exposed to ultra-violet light having a wave length of 2,537 A for suflicient time, about 2 minutes, to kill approximately 99% of the spores. The treated cell suspension was then diluted with distilled water. and a quantity of the diluted suspension was spread over Petri dish plates of nutrient agar, i.e., an aqueous medium containing 2.5% glucose, 4.0% soybean meal, 0.25% sodium chloride, 0.5% distillers solubles and solidified with agar. After incubation until good growth and sporulation occurred, the colonies were transferred to agar slants of the same medium, and, after incubation until good growth and sporulation occurred, sterile water was added to each of the slants, and separate spore suspensions were prepared from each slant. Quantities of each spore suspension were transferred to flasks containing an inoculum medium composed of 1; glucose, 1% enzymatic digest of casein, 1% sodium chloride, 0.6% meat extract, and distilled water to volume. The inoculated flasks were then incubated with constant agitation for 48 hours, and the vegetative growth which developed was used to inoculate flasks of aqueous fermentation medium having the composition: 2.5% glucose, 4.0% soybean meal, 0.25% sodium chloride and 0.5% distillers solubles. These inoculated flasks were then incubated at 28 C. with constant agitation for 4 to 5 days to permit maximum streptomycin .production.

The foregoing mutation and selection procedures were repeated with additional spore suspensions of S. griseus Dulaney L-118 until a superior streptomycin producing mutant was obtained as evidenced by an enhanced yield of streptomycin in the fermentation fiask.

A spore suspension in distilled water'of this superior mutant which was designated O-3 was exposed to soft X-rays until approximately 99% of the spores were killed. This treated suspension was diluted with distilled water, plated on nutrient agar for spore development, and the resulting spores were used to inoculate flasks of inoculum medium and flasks of fermentation medium in accordance with the procedure abovedescribed.

A new and higher yielding mutant thus obtained from O-3 was designated R-315. Spores of the mutant R-315 were similarly treated with X-rays, and from the cultures thus obtained mutant T-535 was selected as a parent for further mutation work. Spores of mutant T-535 were treated with ultra-violet light and mutant V-148 was selected from. the resulting cultures. One of the natural isolates of mutant V-148, i. e., strain X-69, was found to be a superior streptomycin producer and was, in turn, treated with ultra-violet light yielding the superior mutant Z-38, more fully hereinafter referred to as S. griseus, albus mutant (Dulaney Z-38). The following diagram illustrates the genealogy of strain Z-38.

Comparative morphological properties and biochemical reactions as given in Bergeys Manual of Determinative Bacteriology and as observed for S. griseus Dulaney L-118 and S.

griseus, albus mutant (Dulaney Z-38) are tabu- The following examples are presented to illuslated below: trate procedures for the production of strepto- CULTURAL CHARACTERISTICS OF S. GRI SEU S Bergey S ggg g g gg S. griseus L-ns S. priseus Z-38 Filaments Branching, a few spirals Straight, branching, no spirals. Straighlt, branching, occasional spira s. Conidia Rod-shaped to short cylindrical Agrees Agrees.

0.8 x 0.8 to 1.7 microns.

Gelatin stab Grecnish-yellow or cream-colored 16 days-50% of medium liquefied l6days25% oi medium liquefied surface growth, brownish tinge, (color not recorded). (color not recorded). rapid liquefaction.

Synthetic Agar Thin, colorless, spreading, olive Colorless growth no aerial myce- Colorless growth no aerial myocbuti-aerial mycelium thick, ium. ium. powder, water-green.

Starch Agar Thin spreading, Transparent Thin, spreading, not transparent, Thin,spreading, not transparent, cream growth, starch hydrocream growth, starch hydrolyzed. lyzed.

Dextrose Agar Elevated in center, radiate cream- Faint growth-spreading, colorless- Faint growth-spreading, colorless.

colored to orange, crose margin.

Plain Agar Abundant, cream-colored, almost Colorless, transparent growth Colorless, transparent growth.

transparent.

Dextrose broth Abundant, yellowish pellicle with Pellicle ("clot and type not ob- Pellicle (Color and type not obgreenish tinge, much folded. serve served).

Litmus milk Cream colored ring, coagulated Peptonizcd (pH of substrate, and Peptonize'i (pH of substrate, and with rapid pcptonization, becolor and type of surface growth color and type of surface growth coming alkaline. not observed). not observed).

Potato Yellowish, wrinkled Heavy growth, grey; rugose; po- Heavy growth, tan; rugose, po-

teto darkening. tato darkening.

Reduction Nitrites produced from nitmtes.. N itrites produced Nitrites produced.

Pigment Not soluble Not soluI lax-.. Not soluble.

Oxygen tension Aerobic Aerobic Aerobic.

Additional typing tests not listed by Bergey gave the following results:

mycin using the new culture S. griseus, albus mutant (Dulaney Z-38).

Tt s L-118 s E PLEI Spores from nutrient agar slants of the S. Cellulose decomposition. No decomposition... No decomposition. gnseus stralns- 13-118 T and Z 38 previously Ca malate agar Flaint growth Faint growth. referred to were used to inoculate separate flasks Tymsme agar Noappmmgmwmcontaining the following nutrient medium: 1% Phosphate agar spolre-bearilngt hy- Vegetative myceglucose, 1% enzymatic digest of casein, 1% sodip he in c us ers. ium. Soybean agar (Composi- Abundant sporula- Abundant sporulaum chlonde' meat extract dlstlued gizoxbsee page 2, lines tion, grey to greytiyon, spores water to volume. The flasks containing the 1ngreen spores. w rite. 1% Yeast extract. 0.5% Abundant sporula- No sporulntfon. Oculated medium incubated on a rotary sglucose agar}. I Ltign. l 4 P u shaker at 28 C. until good growth occurred.

pore inocu um on ig tsporu ation ractica y nospor- Dim Yeasmeef Agar mom so After 48 hours incubation, this vegetative growth -(N t ee/14); 1 a b W I L h (7 was used to inoculate 250 ml. Erlenmeyer flasks egea ive inocu am A cut 8 of coess t an 1 of on Difco Yeast Beef onies shoiv sporucolonies show contalnlng 40 of the fouowlng medlmn Agar (No. B244). ulation. sporulation. Glucos per cent 2 5 Soybean meal do 4.0 S l Bl k t ses, 533,113 a e W 18 suspend m defied as Sodmm chlorlde c0 0.25 I The composition of Diico Yeast Beef Agar (10. B244) is as iol- Di till dri d s luble do 05 ows:

' Distilled water to volume Grams per pH before sterilization 7.38 Ingredient gifi igg The flasks containing the inoculated medium medium were incubated on a rotary shaker, 220 R. P. M.,

at- 28 C. until maximum streptomycin produc- Bacto Beet Extract 0.15 tion, as measured by the Bacillus subtilus cup Bacto Yeast Extract 0.3 d mew Peptone assay, occurre The results of the comparative Bacto Dextrose 0.1 experiment are given in the following table: Bacto Agar 1.5 NOTE: To this medium was added 1 gram per 100 cc. of gfgfii Diico dehydrated agar to yield a firmer solidified medium. potency 7 m1 I Vegetative inoculum was prepared by inoculating 40 cc. of a Strain after sterile liquid medium described below, in a cotton plugged 250 cc. Erlenmeyer flask and incubating at 27 C. for 20 hours on a rotary shaker. 4days 5days The medium used for vegetative inoculum development had the following composition:

l 635 l. 480 Grams per 2, 000 Ingredient g g ig g medium 1 Streptomycin yield is low for strain L-ll8 due to the fact that the inciilgation temperature is higher than the optimum value for strain g-ZGAmino (enzyme hydrolized casein) 1.0 I EXAMPLE 3 g ifi 3 A 5-l1ter fermentor was charged with 3.2 liters 4 In inoculating with spores, spores from a Blake bottle culture were suspended in sterile water at a suiiicient dilution so that when loop streaked on the test medium, and incubated at 27 C. for about 6 days, separate colonies developed and were examined individually for sporulation.

5 A loop of the vegetative inoculum, prepared as described in note (8) above, was streaked on the test medium. Dilution was such that, after incubation of the test plate for about 6 days at 27 0., separate colonies developed and were examined individually for sporulation.

of a medium containing 4% soybean meal, 0.25% sodium chloride, 0.5% distillers solubles, 2.5% dextrose and distilled water to volume. Approximately 0.5 P. P. M. of cobalt as cobalt nitrate was also added to the medium, and the medium, after sterilization, was inoculated with 5% of a vegetative culture of S. griseus. albus mutant Streptomycin B" color isolated inoculum Ma Timeof gggg} mum $32; volume, meg/ml. hours mg.

L-ll8 1,140 I 1 113 950 Z-38 1,830 1 118 1 The streptomycin yield with strain Z-38 would have been somewhat higher if the optimum temperature of 28.5 0. had been employed in this experiment.

In 15,000 gal. fermentors the time for maximum streptomycin production is 80 hours for L-1l8 and 110 hours for Z-38.

EXAMPLE 3 An inoculum was prepared by propagating spores of S. griseus, albus mutant (Dulaney Z-3'8) in a sterile medium containing 1% dextrose, 1% enzymatic digest of casein, 0.6% meat extract and distilled water to volume, under aerated and agitated conditions at 27 C. for 36to 48 hours until good growth was obtained. One ml. portions of the resulting broth were used to inoculate each of a number of flasks containing the medium above-described, and these cultures were allowed to grow at 27 C. under aerated and agitated conditions for 20 to 24 hours until approximately 5-7 mg./ml. of vegetative growth (dryweight) was obtained and 3-4 mg./ml. of sugar remained. The contents of the flasks were then pooled to provide inoculum for actual streptomycin pro;

' duction.

A number of 5-liter fermentors were charged with 3200 ml. portions of sterile medium containing 3.5% of soybean meal, 2.75% dextrose, distillers dried solubles, 0.2 sodium chloride, 0.4 cc. per 100 cc. of soybean oil and distilled water to volume, which is the preferred medium for Z-38. Each of the fermentors was inoculated with 150 ml. of the prepared inoculum andincubated at a temperature of 285 C. for 112 to 118 hours with mechanical agitation, imparted by an impeller bearing rotary shaft, and aeration under varying conditions. The streptomycin yields obtained with the different conditions of agitation and aeration are tabulated below, the streptomycin yield in each instance being an average of values obtained in 3 or more separate fermentors.

Efiect of power on streptomycin production In comparison, a similar experiment was performed in the same 5-liter fermentors in which vegetative inoculum of culture L-118 was used for fermentation at 27 C. of the following sterile preferred medium: soybean meal 3.0%; dextrose 2.0%, distillers dried solubles 0.75% and sodium chloride 0.25%. Results obtained were as'follows:

As will be noted from the above tables, increasing the horsepower absorbed per gallon of fermenting medium of 0.0024 with culture L-118 does not increase streptomycin yield above 1000 mcgJml. of fermented broth, whereas, with the Z-38 culture such increase in horse power absorbed up to 0.009 horse power per gallon of fermented medium raises the streptomycin yieldup to 2000 meg/ml. of fermenting broth.

EXAIVEPLE 4 The procedure of preparing an inoculum of S. griseus, albus mutant (Dulaney Z-38) and using this inoculum to inoculate 3200 ml. portions of medium in a number of 5-liter fermentors was repeated as described in Example 3. The inoculated mediums were then incubated at the different temperatures as indicated with constant mechanical agitation and aeration until maximum streptomycin production was obtained. Agitation in each instance was supplied by means of 2 turbo impellers revolving at such a rate that the total power absorbed was 0.0091 HP./gal. The streptomycin yield at the different incubation temperatures employed is indicated in the following tabulation, the values in each instance representing an average of two or more separate fermentations.

- Time for Max- Temperature, gggfi imum C. me Streptomycin Yield, hours Additional experiments have indicated that the optimum incubation temperature with the organism S. griseus, albus mutant (Dulaney Z-38) is 285 C.

EXAMPLE 5 in each instance being provided by two turbo impellers revolving at such a rate that the total power absorbed was 0.0091 HP./gal. The fermentations were conducted as two separate experiments, one to determine the comparative effectiveness of pH adjustment before and after sterilization, and the other to determine the optimum pH adjustment before sterilization. The streptomycin yields obtained in these experiments are tabulated below, the values in each instance representing an average of two or more separate fermentations.

Eflect of pH adjustment before and after sterilization Time for Max- Initial Streptomycin pH of pH Adjustment gg i Yield, Sample Yie d, hours mcg./ml.

6. 6 7.0 before sterilization U2 1, 769 6. 8 7.0 after sterilization 118 l. 555 6. 4 6.5 after sterilization 104 l, 378 6. 2 6.0 after sterilization 104 1. 266

Efiect of difierent pH adjustment before and after steri ization Time for Initial pH ad ustment ml. 30? Streptomycin pH of NaOH added before ster g gg Yield Sample ilization Yiefil mcgJml.

6. 65 3.2 (pH approx. 7.0). 112 l, 913 6. 33 2.2 (pH less than 7.0)- 118 l, 859 6. 5 4.2 (pH greater than 7.0)- 96 l, 799

From the foregoing tabulations, it is evident that better results are obtained when the pH is adjusted prior to sterilization and that optimum results are obtained when the pH is adjusted to approximately 7.0 priorto sterilization.

From the foregoing examples, it is evident that 8 the following criteria, in addition to the typing test results already set forth, can be used to distinguish S. lgriseus, albus mutant (Dulaney Z-38) from its parent S. grz'seus Dulaney L-ll8:

Z-38 L-1l8 Optimumfermentationtemperature. 28.5C 27 C. Fermentation time for maximum 110 hours hours streptomycin production. Optimum pH of medium:

Before sterilization 7.0.... 6.2-6.4. Aftersterilization 6.5-6.6..." 6.1-6.3. Minimum agitation power absorp- 0.009 0.0024.

tion for maximum streptomycin production HP/gal. fermenting aerated medium. Approximate maximum yield of 2,000 1,000.

streptomycinmcg. of streptomycin per ml. of fermented broth. Vitamin B potency of fermented none 950 mg. er 1,000 broth. gal. of roth.

REFERENCES CITED The following references are of record in the file of this patent:

UNITED STATES PATENTS Name Date Demerec July 27, 1948 Number 

